[unreadable] Research in our laboratory has led to the synthesis of a novel protein crosslinking reagent, triglycidyl amine (TGA), for preparing calcification resistant heart valve bioprostheses. The overall hypothesis of this proposal is that TGA crosslinking and related reactions, irMuding those with bisphosphonates, enhance biocompatibilitv, improve biomechanics, and inhibit bioprosthetic heart valve degeneration due to maior modifications of the structural proteins of the extracellular matrix (ECM), and cellular interactions with the TGA crosslinked ECM. Aim 1. Mechanisms of TGA crosslinking: The focus here will be on TGA chemistry and biomaterial interactions with studies of reaction conditions, crosslink formation, and resultant biomechanical effects. We will also study the, reactions, reaction kinetics, and net effects on crosslinking and biomechanics involving TGA and 2,2 mercaptoeothylamino-ethylidene-1, 1-bisphosphonate (MABP), a bisphosphonate custom synthesized by our group that can be covalently linked to TGA treated substrates to prevent both bioprosthetic cusp and aortic wall calcification (see Progress Report). Aim 2. To study the cellular and ECM mechanisms responsible for TGA crosslinking conferring resistance to calcification: We will investigate TGA and TGA plus MABP inhibition of calcification as this relates to cellular interactions with the TGA-crosslinked ECM. We will focus on TGA-related up-regulation of osteopontin (OPN), an ECM protein associated with calcification inhibition, and down-regulation of tenascin-C (TNC), an ECM protein associated with heart valve calcification. We will use cell culture studies with sheep aortic valve interstitial cells (SAVIC) to model cell-ECM interactions comparing TGA with and without MABP to control substrates. Aim 3. To investigate in vivo the mechanisms responsible for TGA-crosslinking plus MABP inhibiting bioprosthetic calcification, enhancing biocompatibilitv, and improving biomechanical interactions: Progress thus far in both rat subdermal and sheep circulatory studies has demonstrated TGA crosslinking confers inhibition ofbioprosthetic cusp calcification, and combined with MABP also results in inhibition ofbioprosthetic aortic wall calcification (Progress Report). We will investigate the mechanisms responsible for TGA-induced ECM structural protein modifications with resulting cellular ECM interactions that lead to upregulation of OPN, with associated calcification inhibition, biocompatibility, and enhanced biomechanical properties. [unreadable] [unreadable]